Enzymatic method for detecting coliform bacteria or E. coli

ABSTRACT

A two stage enzymatic method for the detection of coliform bacteria or E. coli wherein bacteria are concentrated on a membrane filter. This filter is placed on a growth medium containing nutrients, including preferably minerals, a protein hydrolysate and a sugar, preferably maltose or a polyalcohol, preferably mannitol, an inducer of a marker enzyme, in particular β-galactosidase or β-glucuronidase and inhibitors of the growth of competing bacteria. After a preincubation step, the filter is placed on an assay medium containing a fluorogenic or chemiluminogenic enzyme substrate and a membrane permeabilizer. The membrane filter and the assay medium are incubated to allow cleavage of the enzyme substrate producing fluorescent or chemiluminescent microcolonies on the membrane filter after triggering of light emission.

RELATED APPLICATIONS

This application claims the priority of PCT Application No.PCT/BE95/00102/, filed Nov. 7, 1995, and PCT Application No.PCT/BE94/00083, filed Nov. 7, 1994, which are incorporated herein byreference.

RELATED APPLICATIONS

This application claims the priority of PCT Application No.PCT/BE95/00102/, filed Nov. 7, 1995, and PCT Application No.PCT/BE94/00083, filed Nov. 7, 1994, which are incorporated herein byreference.

The present invention relates to an enzymatic method for detectingcoliform bacteria, in particular total coliform bacteria faecal coliformbacteria or E. coli, in a liquid or liquefied sample, for exampledrinking or recreational water, comprising the steps of:

a) concentrating the bacteria on a membrane filter;

b) placing the membrane filter and the bacteria concentrated thereon ona growth medium containing nutrients to support propagation of thebacteria and an inducer for inducing a marker enzyme in the course oftheir growth and metabolism;

c) preincubating the membrane filter and the bacteria concentratedthereon to form microcolonies of these bacteria on the membrane filterand to produce said marker enzyme; and

d) making the microcolonies visible by means of luminescence.

Coliform bacteria are indicators of the sanitary quality of water andfood. Total coliforms (TC) in water originate from soil or organicvegetal material. Faecal (thermotolerant) coliforms (FC) and E. coli inparticular inhabit the intestine of humans and animals and areindicators of faecal pollution.

Traditional processes for detecting coliforms and E. coli by membranefiltration are based on lactose fermentation in conjunction withconfirmatory tests and require 48 to 96 hours to complete. A procedureis conventionally considered to be rapid if it takes 24 hours or less toperform. However, a 24 hours method is still not rapid enough to be usedfor the analysis of drinking water in emergency situations, e.g. afterbreakdowns in the water supply or construction works to the distributionsystem. In those cases, the detection of at least 1 coliform bacteriumper 100 ml of water should be feasible within the ordinary work shift of8 hours and preferably in maximum 7 hours to demonstrate the potabilityof the water and, hence to avoid unnecessary warnings to the publicabout the contrary.

Existing rapid (24 hours) membrane filtration methods for the detectionof coliform bacteria, in particular TC and E. coli rely on thedemonstration of the activity of 2 specific marker enzymes in thebacterial colonies, i.e. β-galactosidase and β-glucuronidase,respectively, which the bacteria produce as they grow and metabolize.The presence of these enzymes is revealed by the ability of the bacteriapresent on the membrane filter to cleave chromogenic substrates added tothe growth medium such as5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) forβ-galactosidase and 5bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc)for β-glucuronidase. The chromogenic substrates themselves are notcolored so that the detection of colored colonies on the membrane filterindicates the presence of the enzyme and, hence, of the bacteria. Seee.g. Manafi and Kneifel, Zentralbl. Hyg. 189:225-234 (1989), Brenner etal., Appl. Environ. Microbiol. 59:3534-3544 (1993) and Frampton andRestaino, J. Appl. Bacteriol. 74:223-233 (1993).

Similarly, fluorogenic substrates, e.g.4-methylumbelliferyl-β-D-galactopyranoside (MU-gal) or4-methylumbelliferyl-β-D-glucuronide (MUG) added to the growth mediumcan be cleaved by bacterial β-galactosidase and β-glucuronidase,respectively, to yield a fluorescent product, 4-methylumbelliferone(4-MU). The fluorogenic substrates themselves do not fluoresce so thatthe detection of fluorescent colonies on the membrane filter indicatesthe presence of the enzyme and, hence, of the bacteria. Currently, themost rapid fluorescent method to detect TC on a membrane filter usingMU-gal as a substrate for β-galactosidase takes 16-24 hours to complete(Brenner, cited above). For E.coli the minimal detection time obtainedby using MUG as a substrate for β-glucuronidase is 7.5 hours (Sarhan andFoster, J. Appl. Bacterial. 70:394-400 1991)). The Berg et al. U.S.patent and scientific publication disclose a method to detect faecal(thermotolerant) coliforms on a membrane filter using an agar growthmedium containing MU-gal as a substrate for β-galactosidase and anincubation temperature of 41.5° C., in a time period of 6 hours (Berg etal., U.S. Pat. No. 5,292,644 and Appl. Environ. Microbiol. 54:2118-2122(1988)). However, the time to detect total coliforms which grow at35°-37° C. and possess lower β-galactosidase activity than thethermotolerant coliforms exceeds 8 hours. E. coli cannot be detectedspecifically using this method.

SUMMARY OF THE INVENTION

The object of the present invention is therefore to provide a membranefiltration method which allows to detect coliform bacteria, inparticular FC but especially also TC and E. coli in a liquid orliquefied sample in a shorter time, for TC for example within adetection time of 7 hours.

To this end, themethod according to the present invention ischaracterized in that said microcolonies are made visible by an enzymeassay comprising:

removing the membrane filter from the growth medium after saidpreincubation step (c);

treating the microcolonies on the membrane filter with a membranepermeabilizer and contacting them with a chemiluminogenic or fluorogenicsubstrate for said marker enzyme;

incubating the microcolonies to allow cleavage of said substrate so asto produce a chemiluminescent or fluorescent product; and

triggering light emission from the chemiluminescent or fluorescentproduct produced by the microcolonies.

The use of a two-stage approach in which the enzyme assay step isseparated from a so-called preincubation step meant to propagate thebacteria and to induce the marker enzyme, allows to add a membranepermeabilizer and to avoid competition for the enzyme between thesubstrate and the inducer. The rationale for including a membranepermeabilizer in the assay is to promote the rapid uptake of thesubstrate by the bacteria. Use is made in particular of membranepermeabilizers that disrupt both the outer and the cytoplasmic membraneof coliforms and E. coli, for example polymyxin B or colistin, or amixture of one of these with lysozyme. In the known one-stage methods,for example the method disclosed by Berg et al. in U.S. Pat. No.5,292,644, such membrane permeabilizers can not be used since they arebactericidal and can thus not be added to the growth medium.

A two-stage method has already been disclosed by the present inventor,i.e. by Nelis et al. in Proceedings of the Water Quality TechnologyConference, (AWWA), Miami, Fla, 7-11 Nov., 1993, pp. 1663-1673 In thisknown method, the bacteria concentrated on the filter are howevertransferred into a liquid growth medium, the filter itself being removedso that no microcolonies are formed in the medium. Instead, ahomogeneous mixture of bacteria is obtained from which the overall lightemission is measured by means of a luminometer or a fluorometer. It wasfound now, however, that in such liquid detection technique non-coliformbacteria which contain β-galactosidase or β-glucuronidase may ofteninterfere. Although the growth of such non-coliform bacteria can beinhibited, the amount of marker enzyme present in these non-coliformbacteria influences the measurement when large numbers of them arepresent in the sample. In the method according to the present invention,such an interference is excluded since the non-coliform bacteria can beprevented from forming microcolonies, even if they are present in a muchlarger number than the coliform bacteria.

In a particular embodiment of the present invention, use is made of amembrane filter which is more hydrophobic than generally used celluloseester filters, use being in particular made of a membrane filter made ofpolyvinylidenedifluoride (PVDF).

It was found that the use of such hydrophobic membrane filters resultsin intensified light emission by the bacterial microcolonies.

In a preferred embodiment of the present invention, said inducer isselected from the group comprising isopropyl-β-D-thiogalactopyranosidefor β-galactosidase and isopropyl-β-D-thioglucuronide andpnitrophenyl-β-D-alucuronide for β-glucuronidase.

These inducers were found to be more efficient than the inducers used intraditional media.

In a further preferred embodiment of the present invention, use is madeas said growth medium of a medium containing mineral nutrients, aprotein hydrolysate, in particular tryptone, and a sugar, preferablymaltose, or a polyalcohol, preferably mannitol.

The use of such a growth medium in the preincubation step combines theproperties of efficient growth promotion, good recovery of stressedcoliforms/E. coli on one hand with a low luminescent background andminimal effects of quenching of light emission on the other hand.

In a still further preferred embodiment of the present invention, use ismade of fluorogenic substrates different from the above mentioned MU-galand MUG, in particular of4-trifluoromethylumbelliferyl-β-D-galactopyranoside (TFMU-gal) or4-trifluoromethylumbelliferyl-β-D-glucuronide (TFMUG), but preference isgiven to chemiluminogenic substrates. The latter have not been appliedso far for the detection of bacterial colonies grown on a membranefilter but yield more sensitivity than the presently used substrates.

The present invention also relates to a growth medium for use in themethod according to the invention, which medium is characterized in thatit contains mineral nutrients, a protein hydrolysate, in particulartryptone, an inducer for inducing a marker enzyme in the course ofgrowth and metabolism of coliform bacteria, in particularβ-galactosidase or β-glucuronidase, and a sugar, preferably maltose or apolyalcohol, preferably mannitol.

Before giving a more detailed description of the invention, the meaningsof some specific terms used in this description are first of all givenhereinafter:

The term total coliforms (TC) refers to bacteria belonging to either offour genera, i.e. Escherichia, Enterobacter, Klebsiella or Citrobacter,and possessing the enzyme β-galactosidase.

The term faecal coliforms (FC) refers to (thermotolerant) bacteriabelonging to the group of the coliforms and inhabiting the intestine ofhumans and animals. These faecal coliforms are indicators of faecalpollution and posses also the enzyme β-galactosidase, the particularspecies E. coli possessing further the enzyme β-glucuronidase. Detectionof the faecal coliforms can be done by incubating them at a highertemperature (41.5°-44° C.) than the temperature used for detecting totalcoliforms (about 35°-37° C.).

The term preincubation refers to a step in the method of this inventionin which a membrane covered with one or more bacteria, the latteroriginating from a filtered sample, is placed on a growth medium andkept at a certain temperature for a given time in order to propagate thebacteria and to induce the marker enzyme.

The term membrane permeabilizer refers to any compound capable ofdisrupting both the outer and the cytoplasmic membrane of bacteria so asto facilitate the uptake of chemicals.

The term enzyme assay refers to a step distinct from the growth step inthe method of this invention in which a substrate is cleaved by a markerenzyme, in particular β-galactosidase or β-glucuronidase, present in thebacteria, the cleavage product then being determined by virtue of thelight it emits after photochemical or chemical excitation.

The term luminescence refers to fluorescence or chemiluminescence.

The term fluorescence refers to a physicochemical process in which amolecule emits light of a certain wavelength after photochemicalexcitation, i.e. with light of a shorter wavelength.

The term chemiluminescence refers to a physicochemical process in whicha molecule emits light after chemical excitation with a formulationtermed "accelerator".

The term fluorogenic substrate refers to a compound which itself isnon-fluorescent but which contains a structural part, i.e. the so-calledfluorescent product, that does emit light when liberated from the parentcompound and photochemically excited.

The term chemiluminogenic substrate refers to a compound which itself isnot chemiluminescent but which contains a structural part, i.e. thechemiluminescent product, that does emit light when liberated from theparent compound and chemically excited.

The sample to be analyzed is liquid or liquefied and is suspected ofcontaining at least 1 TC, 1 FC or 1 E. coli/100 ml. Typical samples towhich the method of the invention can be applied include drinking water,bathing water or liquid extracts of foods or pharmaceuticals.

The first step of a particular embodiment of the method according tothis invention consists of concentrating the bacteria present in forexample a 100 ml liquid or liquefied sample on a filter, moreparticularly on a bacterial membrane filter with a pore size of 0.22μm-0.45 μm and a diameter of for example 47 mm. The membrane filter ispreferably made of polyvinylidene difluoride (PVDF) (Durapore®,obtainable from Millipore, Bedford, Mass.) because this material hasbeen found to enhance the final fluorescence or chemiluminescence of thebacterial microcolonies. Other useful materials includepolytetrafluoroethylene, polycarbonate, nylon or cellulose esters,particularly cellulose nitrate.

The second step is placing the membrane filter containing the bacteriaon a solid, semi-solid or liquid growth medium in a 55 mm petri dishcontaining agar or an absorbent cellulose pad as an inert matrix. Thismedium includes in particular a protein hydrolysate, preferably tryptone(for example 0.5%), yeast extract (for example 0.3%), monoammoniumphosphate (for example 0.2%), magnesium sulfate (for example 0.005%),dipotassium hydrogen phosphate (for example 0.1%) and a sugar,preferably maltose (for example 0.5%) or a polyalcohol, preferablymannitol (for example 0.5%). This medium is called hereinafter theImproved Luminescence Medium (ILM). A further improved medium canhowever be obtained by the omission of yeast-extract from the ILMresulting in more intensely luminescent colonies on the membrane filter.The ILM growth medium further contains sodium dodecyl sulfate (forexample 0.01%), bile salts (for example 0.01%) and the antibioticcefsulodin (for example 0.001%) as inhibitors of competing non-targetbacteria. It further contains a compound to induce one of the saidmarker enzymes, that is for example isopropyl-β-D-thiogalactopyranoside(for example 0.001%) or melibiose (for example 0.01%) an inducer ofβ-galactosidase and p-nitrophenyl-β-D-glucuronide (for example 0.05%),isopropyl-β-D-thioglucuronide (for example 0.001%),o-nitrophenyl-β-D-glucuronide (for example 0.05%) ormethyl-β-D-glucuronide (for example 0.005%) to induce β-glucuronidase.The ILM used in the method according to this invention, and especiallythe ILM without yeast extract, yields considerably more intensefluorescent and chemiluminescent microcolonies after a given time than avariety of other selective media for TC and E. coli, including m Endoagar LES, MacConkey agar, m TEC agar, m FC agar, EC agar, LaurylTryptose agar, Tryptone Bile agar, m T7 agar, Lauryl Sulphate agar, theMUG-7 medium as described by Sarhan and Foster (cited above) and themedium disclosed in the Berg et al. U.S. patent (cited above).

The third step is the so-called preincubation and consists of incubatingthe membrane filter and the growth medium at a suitable temperature (forexample 35°-370° C. for total coliforms or 41.5°-44° C. for faecalcoliforms and especially for E. coli), preferably in a water bath, for asuitable time, to allow the bacteria to multiply and to producesufficient marker enzyme detectable by fluorescence orchemiluminescence.The required preincubation time is usually comprisedbetween 4 and 6.5 hours depending on whether a fluorogenic or achemiluminogenic substrate is used.

The fourth step consists of removing the membrane filter from the growthmedium and placing it on an absorbent cellullose pad impregnated with amedium to assay the marker enzyme. This assay medium contains inparticular a fluorogenic substrate for either of the two marker enzymes,that is preferably TFMU-gal (λexc 394, λem 489 nm) (β-galactosidase) orTFMUG (λexc 394, λem 489 nm) (β-glucuronidase). The common fluorogenicsubstrates MU-Gal (β-galactosidase) or MUG (β-glucuronidase) can also beused but yield a lower sensitivity. A disandvantage of the latter twocompounds is that they require spraying of the membrane filter withsodium hydroxide to yield optimal fluorescence. Other analogues ofMU-gal that could also be considered as substrates for β-galactosidase,including 3-acetyl-7-(β-D-galactopyranosyloxy)coumarin (λexc 420, λem459 nm), 3-(2-benzoxazolyl)-7-(β-D-galactopyranosyloxy)coumarin and1-(β-D-galactopyranosyloxy)-pyrene-3,6,8-tris-(dimethyl-sulfonamide)(λexc. 495 nm, λem. 550 nm at pH 9) (see Koller et al., Appl. Fluoresc.Technol. 1,15-16 (1989)) are in principle more sensitive and specificthan MU-gal itself as their wavelengths of excitation and emission haveshifted to higher values and/or because they have increased molarabsorption coefficients. Non-umbelliferyl fluorogenic galactopyranosidesand glucuronides can in principle also be used, for example fluoresceindi-β-D-galactopyranoside or fluorescein-di-β-D-glucuronide andderivatives thereof such as C₁₂ -fluorescein-di-β-D-galactopyranoside orC₁₂ -fluorescein-di-β-D-glucuronide (ImaGene Green, Molecular Probes,Eugene, OR) or resorufin-β-D-galactopyranoside orresorufin-β-D-glucuronide (ImaGene Red, Molecular Probes). A stillhigher intrinsic sensitivity is associated with the chemiluminogenicAMPGD (3-(4-methoxyspiro (1,2-dioxetane-3,2'-tricyclo 3.3. 1.1³.7!decan!-4-yl)phenyl)-β-D-galactopyranoside) or derivatives thereof, inparticular chloro derivatives (for example Galacton® (a mono-chloroderivative of AMPGD), Galacton-Plus® (a di-chloro derivative of AMPGD)(β-galactosidase) available from Tropix, Inc., Bedford, Mass.) andGlucuron® (3-(4-methoxyspiro 1,2-dioxetane-3,2'-(5'-chloro)-tricyclo3.3.1.1³.7 !decan!4-yl)phenyl)-β-D-glucuronide) or derivatives thereof(β-glucuronidase) (Tropix Inc.), chemiluminescence in general beingsuperior in sensitivity to fluorescence by the order of magnitude. AMPGDand Glucuron have been used as substrates in gene reporter assays. Seee.g. Jain et al., Anal. Biochem. 199:119-124 (1991) and Bronstein etal., Anal. Biochem. 219:169-181 (1994). Furthermore, the assay mediumwill contain a membrane permeabilizer, preferably polymyxin B sulfate orcolistin methanesulfonate, or a mixture of one of these with lysozyme,and buffering substances to adjust z e pH to 7.3. The absorbent padtogether with the membrane filter is incubated at a suitabletemperature, preferably 44° C. in a hot air incubator for a sufficienttime, usually between 15 and 60 minutes to allow the uptake of thesubstrate by the bacteria and its cleavage by the marker enzyme.

The fifth step depends on whether the used substrate was fluorogenic orchemiluminogenic. With a fluorogenic substrate the fifth step comprisesirradiating the bacterial microcolonies formed on the membrane filterduring the preincubation with light of a wavelength close to theexcitation wavelength of the 4-methylumbelliferone, the umbelliferoneanalog or the fluorescent cleavage product in general, so as to causethe microcolonies to fluoresce. The fluorescent microcolonies can bedetected and counted under a UV lamp emitting light of the saidwavelength, for example 366 nm, or by using an instrumental technique,e.g. a CCD camera connected to a computer to process the images. When achemiluminogenic substrate is used the membrane filter is sprayed withan accelerator so as to cause the microcolonies on the filter to becomechemiluminescent. This step is preceded by drying the said filter fore.g. 10 min in a hot air incubator at a suitable temperature, forexample 60° C. The accelerator may be composed of two components, thefirst one being a cationic polymer, either or not in combination with afluorescent dye, e.g. sodium fluorescein and commercially available fromTropix, Inc. as Emerald®, Emerald II®, Sapphire®, Sapphire II®,Nitro-Block® or Ruby®. The second component is an alkalinizing agentsuch as sodium hydroxide. According to the present invention it wasfound that the latter can be replaced by a dilute solution, for example0.5M of an organic base, preferably piperidine, which results inenhanced sensitivity. This new accelerator will further be called"Modified Luminescence Amplifier Material solution" (modifiedLAM-solution). Alternatively, instead of an accelerator prepared bymixing the two components a commercial mixture can be used such asGalacto-Light® or GUS-Light® (Tropix Inc.). As will be demonstratedhereinafter, it was found surprisingly that on PVDF membrane filters acomparable sensitivity can be obtained by using an accelerator fromwhich the first component has been omitted, that is one consisting of0.5M piperidine only. This accelerator will further be called"Alternative Accelerator". The luminescent microcolonies can be detectedby exposing the membrane filter to X-ray film for e.g. 60 minutes, ahigh sensitivity Polaroid® film in an ECL camera dedicated tochemiluminescence or by using an instrumental technique, e.g. a CCDcamera connected to a computer for image processing.

Further particularities of this invention will become apparent from thefollowing description of six examples. However, this description is notintended to limit the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of the appearance of fluorescent microcolonies ofTC and E. coli grown on a membrane filter.

FIG. 2 is a graph correlating counts obtained with the 7-hourfluorescent process for TC on a membrane filter with those obtained on astandard agar medium for the detection of TC.

FIG. 3 is a graph correlating counts obtained with the 7-hourfluorescent process for E. coli on a membrane filter with those obtainedon a standard agar medium for the detection of E. coli.

FIG. 4 is a photograph of X-ray film showing chemiluminescentmicrocolonies of TC and E. coli grown on a membrane filter.

FIG. 5 is a graph correlating counts obtained with the 6.5-hourchemiluminescent process for TC on a membrane filter with those obtainedon a standard agar medium for the detection of TC.

FIG. 6 is a graph correlating counts obtained with the 6.5-hourchemiluminescent process for E. coli on a membrane filter with thoseobtained on a standard agar medium for the detection of E. coli.

FIG. 7 is a photograph of X-ray film showing the increased intensity ofthe microcolonies of TC on polyvinylidenedifluoride (PVDF) membranefilters compared to the intensity on membrane filters made of mixedcellulose esters.

FIG. 8 is a photograph of X-ray film showing the comparable sensitivityobtained for TC when said PVDF filter is sprayed with an acceleratorthat contains only piperidine (Alternative Accelerator), with referenceto spraying with a modified LAM-solution.

FIG. 9 is a picture showing the superiority of the ILM relative to other(commercial) selective media for the detection of TC and/or E. coli, inconnection with the enumeration of E. coli on a membrane filter based onchemiluminometric measurement of β-galactosidase activity.

FIG. 10 is a picture showing the superiority of the ILM relative toother selective media for the detection of TC and/or E. coli, inconnection with the enumeration of E. coli on a membrane filter based onchemiluminometric measurement of β-glucuronidase activity.

FIG. 11 is a picture showing the improved chemiluminescent responseobtained by using the ILM without yeast extract, relative to thecomplete ILM, for preincubation.

DETAILED DESCRIPTION OF THE DRAWINGS EXAMPLE 1

This example demonstrates the detection and counting of TC and E. colion a membrane filter in connection with the use of fluorogenicsubstrates for β-galactosidase or β-glucuronidase, respectively.

Materials:

Membrane filters: 47 mm membrane filters consisting of PVDF (Durapore®,Millipore, Bedford, Mass.), with a pore size of 0.45 μm.

Growth/induction medium: ILM, containing 0.5% maltose, 0.5% tryptone,0.2% monoammonium phosphate, 0.5% sodium chloride, 0.05% magnesiumsulfate, 0.01% sodium dodecyl sulfate, 0.3% yeast extract, 0.01% bilesalts, 0.001% cefsulodin, 0.1% dipotassium phosphate and 1% agar (pH7.3). This medium further either contains 0.001%isopropyl-β-D-thiogalactopyranoside to induce β-galactosidase in TC or0.05% p-nitrophenyl-β-D-glucuronide to induce β-glucuronidase in E.coli. Buffered assay mixture: 0.1 mol/liter sodium phosphate buffersolution (pH 7.3) containing either4-trifluoromethylumbelliferyl-β-D-galactopyranoside (TFMU-gal)(substrate for β-galactosidase) or4-trifluoromethylumbelliferyl-β-D-glucuronide (TFMUG) (substrate forβ-glucuronidase) and polymyxin B sulfate (Sigma, 7730 IU/ml). The assaymixture also contains 1 mM magnesium chloride.

Procedure:

100 ml of a water sample containing TC or E. coli was filteredthrow,=the membrane filter. The filter was aseptically placed on thegrowth/induction medium (ILM) and incubated in a water bath at either37° C. (for TC) or 41.5° C. (for E. coli). After incubation for 6.5hours, the membrane filters were transferred to a second petri dishcontaining an absorbent cellulose pad impregnated with 1.8 ml of thebuffered assay mixture and incubated for another 30 min at 44° C.Following incubation, the fluorescent microcolonies were counted withthe aid of a longwave U; lamp (emitting light at 366 nm)

Results:

FIG. 1 shows an example of the appearance of the fluorescentmicrocolonies of TC (left) and E.coli (right) after 6.5 hours ofincubation and 30 minutes of enzyme assay. FIG. 2 compares the number ofmicrocolonies of TC counted on the basis of fluorescence with thatobtained on a conventional selective agar medium for TC, i.e. m Endoagar LES. FIG. 3 compares the number of microcolonies of E. coli countedon the basis of fluorescence with that obtained by counting on aconventional selective agar medium for E.coli, i.e. m FC agar. Theplotted lines represent the ideal correlation curves, and are not fittedto the data. The correlation coefficient between fluorometric countingand counting on a conventional medium was 0.844 for the detection of E.coli and 0.697 for the detection of TC (n=13).

Conclusion:

The method is capable of detecting and enumerating microcolonies of TCand E. coli on the basis of fluorescence within a total time period of 7hours. The agreement with standard methods was satisfactory.

EXAMPLE 2

This example demonstrates the detection and enumeration of TC and E.coli on a membrane filter in connection with the use of chemiluminogenicsubstrates for β-galactosidase and β-glucuronidase, respectively.

Materials:

Membrane filters: as described in Example 1. Growth/induction medium:ILM, as described in Example 1.

Buffered assay mixture: 0.1 mol/liter sodium phosphate buffer solution(pH 7.3) containing either Galacton-Plus®, 9 μmol/liter (substrate forβ-galactosidase) or Glucuron®, 25 μmol/liter (substrate forβ-glucuronidase)(both substrates from Tropix, Inc.) and polymyxin Bsulfate (Sigma, 7730 IU/ml). The assay mixture also contains 1 mMmagnesium chloride.

Modified Luminescence Amplifier Material-solution (modifiedLAM-solution): Emerald II® (Tropix Inc.), 1.6 μg/ml in aqueous 0.5mol/liter piperidine solution.

Procedure:

100 ml of a water sample containing TC or E. coli was filtered throughthe membrane filter. The filter was aseptically placed on thegrowth/induction medium (ILM) and incubated in a water bath at either37° C. (for TC) or 41.5° C. (for E. coli). After incubation for 6 hours,the membrane filters were transferred to a second petri dish containingan absorbent cellulose pad impregnated with 1.8 ml of the buffered assaymixture and incubated for another 30 min at 44° C. Following incubation,the membranes were sprayed with the modified LAM-solution and thechemiluminescent microcolonies were visualised with the aid of X-rayfilm. To this end the membrane filters were contacted for 1 hour with aAgf a Curix RP1 X-ray film wrapped in polyethylene foil. Afterdevelopment, the luminescent microcolonies appeared as black dots on atransparent background and were counted.

Results:

FIG. 4 shows an example of the appearance of the microcolonies of TC(left) and E. coli (right) on X-ray film after 6 hours of incubation and30 minutes of enzyme assay. FIG. 5 compares the numbers of microcoloniesof TC counted on the basis of chemiluminescence with that obtained bycounting conventional selective agar medium for TC, i.e. m Endo agarLES. FIG. 6 compares the numbers of microcolonies of E. coli obtained bycounting on the basis of chemiluminescence with that obtained bycounting on a conventional selective agar medium for E.coli, i.e. m FCagar. The plotted lines represent the ideal correlation curve, and arenot fitted to the data. The correlation coefficient of thechemiluminometric direct counting process compared with conventionalmedia was 0.836 for the detection of E. coli and 0.650 for the detectionof TC (n=13).

Conclusion:

The method is capable of detecting and enumerating microcolonies of TCor E. coli on the basis of chemiluminescence within a total time periodof 6.5 hours, in particular when an instrumental technique is used, forexample with a CCD camera connected to a computer for image processinginstead of the X-ray film. The agreement with standard methods wassatisfactory.

EXAMPLE 3

This example demonstrates the superiority of polyvinylidenedifluoride(PVDF) filters over conventional filters made of mixed cellulose estersfor the enumeration of TC on a membrane filter, based onchemiluminescence.

Materials:

Membrane filters: 47 mm membrane filters consisting of PVDF (Durapore®,Millipore, Bedford, Mass.), with a pore size of 0.45 μm.

47 mm membrane filters made of mixed cellulose esters, with a pore sizeof 0.45 μm (Millipore).

Growth/induction medium: ILM, containing 0.5% maltose, 0.5% tryptone,0.2% monoammonium phosphate, 0.5% sodium chloride, 0.05% magnesiumsulfate, 0.01% sodium dodecyl sulfate, 0.3% yeast extract, 0.01% bilesalts, 0.001% cefsulodin, 0.1% dipotassium phosphate and 1% agar (pH7.3). This medium further contains 0.001%isopropyl-β-D-thiogalactopyranoside to induce β-galactosidase in TC.

Buffered assay mixture: 0.1 mol/liter sodium phosphate buffer solution(pH 7.3) containing Galacton-Plus® (Tropix, Inc.), 9 μmol/liter andpolymyxin B sulfate (Sigma, 7730 IU/ml). The assay mixture also contains1 mM magnesium chloride.

Modified Luminescence Amplifier Material-solution (modifiedLM-solution): as described in Example 2.

Procedure:

100 ml of a water sample containing TC was filtered through the membranefilter. The filter was aseptically placed on the growth/induction medium(ILM) and incubated in a water bath at 37° C. After incubation for 6hours, the membrane filter was transferred to a second petri dishcontaining an absorbent cellulose pad impregnated with 1.8 ml of thebuffered assay mixture and incubated for another 30 min at 44° C.Following incubation, the membrane was sprayed with the modifiedLAM-solution and the chemiluminescent microcolonies were then visualisedwith the aid of X-ray film, as described in Example 2.

Results:

FIG. 7 is a picture showing the increased intensity of the microcoloniesof TC on PVDF membrane filters relative to the intensity obtained onmembrane filters made of mixed cellulose esters.

Conclusion:

Membrane filters made of PVDF yield more intensely chemiluminescentmicrocolonies of coliforms than filters made of mixed cellulose esters,thus improving the detectability of the bacteria.

EXAMPLE 4

This example demonstrates that the visualisation of total coliforms on amembrane filter based on chemiluminescence can be performed with adilute solution of an organic base without the addition of a cationicpolymer and a fluorescent dye when hydrophobic membrane filters, inparticular PVDF (Durapore®) are used to concentrate the bacteria presentin the sample.

Materials:

Membrane filters: as described in example 1. Growth/induction mediumILM, as described in Example 3.

Buffered assay mixture: as described in Example 3. Modified LuminescenceAmplifier Material-solution (modified LAM-solution): as described inExample 2. Accelerator without cationic polymer and fluorescent dye:aqueous 0.5M piperidine (called "Alternative Accelerator")

Procedure:

As described in Example 3, except that following incubation, themembrane was sprayed either with the modified LAM-solution or theAlternative Accelerator. The chemiluminescent microcolonies were thencounted with the aid of X-ray film, as described in Example 2.

Results:

FIG. 8 illustrates the comparable sensitivity obtained by spraying aPVDF filter with an accelerator that contains only piperidine(Alternative Accelerator, right), with reference to spraying with anaccelerator containing piperidine and a Luminescence Amplifying Material(left).

Conclusion:

The hydrophobic environment, created by the PVDF filter protects theenzymatic cleavage product, which makes the use of a protective cationicpolymer superfluous and, hence, allows the use of an accelerator thatconsists of only piperidine.

EXAMPLE 5

This example demonstrates the superiority of the ILM over conventionalselective media for the detection of TC and/or E. coli, in connectionwith the enumeration of E. coli on a membrane filter, based on thechemiluminometric detection of either β-galactosidase orβ-glucuronidase.

Materials:

Membrane filters: as described in Example 1. Bacterial strain: E. coli,No. 2, isolated from a natural well water. An appropriate dilution of anovernight culture in tryptic soy broth w/o dextrose (DIFCO) was added tosterile distilled water, so as to obtain a concentration ofapproximately 100 CFU/100 ml. Growth/induction media: ILM, containing0.5% maltose, 0.5% a tryptone, 0.2% monoammonium phosphate, 0.5% sodiumchloride, 0.05% magnesium sulfate, 0.01% sodium dodecyl sulfate, 0.3%yeast extract, 0.01% bile salts, 0.001% cefsulodin, 0.1% dipotassiumphosphate and 1% agar (pH 7.3). This medium either contains 0.01%isopropyl-β-D-thiogalactopyranoside to induce β-galactosidase or 0.005%p-nitrophenyl-β-D-glucuronide to induce β-glucuronidase.

Lauryl Tryptose agar: Lauryl Tryptose broth (Difco, Detroit, Mich.) towhich 1% agar was added, as such (β-galactosidase) or supplemented with0.005% p-nitrophenyl-β-D-glucuronide (β-glucuronidase).

EC agar: EC-broth (Difco) to which 1% agar was added, as such(β-galactosidase) or supplemented with 0.005%p-nitrophenyl-β-D-glucuronide (β-glucuronidase).

m T7 agar (Difco), as such (β-galactosidase) or supplemented with 0.005%p-nitrophenyl-β-D-glucuronide (β-glucuronidase)

m TEC agar (Difco), as such (β-galactosidase) or supplemented with0.005% p-nitrophenyl-β-D-glucuronide (β-glucuronidase).

m FC agar (Difco), as such (β-galactosidase) or supplemented with 0.005%p-nitrophenyl-β-D-glucuronide (β-glucuronidase).

MacConkey agar (Difco), as such (β-galactosidase) or supplemented with0.005% p-nitrophenyl-β-D-glucuronide (β-glucuronidase).

m Endo agar LES (Difco), as such (β-galactosidase) or supplemented with0.005% p-nitrophenyl-β-D-glucuronide (β-glucuronidase).

Lauryl Sulphate agar: Lauryl Sulphate broth (Oxoid, Ltd., Basingstoke,U.K.) to which 1% agar was added, as such (β-galactosidase) orsupplemented with 0.005% p-nitrophenyl-β-D-glucuronide(β-glucuronidase).

Tryptone Bile agar: containing 2% tryptone, 0.16% bile salts and 1%agar, to which further 0.01% isopropyl-β-D-thiogalactopyranoside(β-galactosidase) or 0.005% p-nitrophenyl-β-D-glucuronide(β-glucuronidase) was added,

MUG-7 agar: medium as described by Sarhan and Foster (cited above), butwith MUG omitted and 0.01% isopropyl-β-D-thiogalactopyranoside(β-galactosidase) or 0.005% p-nitrophenyl-β-D-glucuronide(β-glucuronidase) added,

medium disclosed in the Berg et al. U.S. patent (cited above), but withMU-gal omitted, as such (β-galactosidase) or supplemented with0.005%p-nitrophenyl-β-D-glucuronide (β-glucuronidase). Buffered assaymixture: as described in Example 2. Modified Luminescence AmplifierMaterial-solution (modified LAM-solution): Sapphire II (Tropix, Inc.)1.6 μg/ml in aqueous 0.5 mol/liter piperidine solution.

Procedure:

As described in Example 2. Except that in addition to the ILM a varietyof other growth/induction media were used.

Results:

FIG. 9 is a picture showing the superiority of the ILM (A) relative toother.(commercial) selective media (B to L) for the detection of TCand/or E. coli, in connection with the enumeration of E. coli on amembrane filter based on chemiluminometric measurement ofβ-galactosidase activity, wherein B=the MUG-7 medium as described bySarhan and Foster (cited above), C=Tryptone Bile agar, D=the mediumdisclosed in the Berg et al. U.S. patent (cited above), E=LaurylTryptose agar, F=EC agar, G=m T7 agar, H=m TEC agar, I=MacConkey agar,J=m FC agar, K=m Endo agar LES and L=Lauryl Sulphate agar.

FIG. 10 is a picture showing the superiority of the ILM (A) relative toother (commercial) selective media (B to L) for the detection of TCand/or E. coli, in connection with the enumeration of E. coli on amembrane filter based on chemiluminometric measurement ofβ-glucuronidase activity, wherein B=the MUG-7 medium as described bySarhan and Foster (cited above), C=Tryptone Bile agar, D=the mediumdisclosed in the Berg et al. U.S. patent (cited above), E=LaurylTryptose agar, F=EC agar, G=m T7 agar, H=m TEC agar, I=MacConkey agar,J=m FC agar, K=m Endo agar LES and L=Lauryl Sulphate agar, all mediasupplemented with 0.005% p-nitrophenyl-β-D-glucuronide.

Conclusion:

The use of the ILM is an important feature for the optimal performanceof the method according to this invention for the detection ofmicrocolonies on the basis of β-galactosidase or of β-glucuronidaseactivity.

EXAMPLE 6

Materials:

Bacterial strain: as described in Example 5. Membrane filters asdescribed in Example 1. Growth/induction medium with yeast extract: ILM,as described in Example 1, containing 0.05%p-nitrophenyl-β-D-glucuronide to induce β-glucuronidase.

Growth/induction medium without yeast extract: the ILM, as described inExample 1 but with omission of the yeast extract and containing 0.05%p-nitrophenyl-β-D-glucuronide to induce β-glucuronidase.

Buffered assay mixture: 0.1 mol/liter sodium phosphate buffer solution(pH 7.3) containing Glucuron® (Tropix, Inc.), 25 μmol/liter andpolymyxin B sulfate (Sigma, 7730 IU/ml). The assay mixture also contains1 mM magnesium chloride.

Modified Luminescence Amplified Material solution (modified LAMsolution): as described in Example 5.

Procedure:

E. coli was detected and counted on a membrane filter based onchemiluminescence, as described in Example 2, using Glucuron as asubstrate but with omission of the yeast extract from the ImprovedLuminescence Medium (ILM).

Result:

FIG. 11 illustrates the enhanced chemiluminescent response for E. coliobtained by using the ILM without yeast extract (right), relative to thecomplete ILM (left), for preincubation.

Conclusion:

The ILM without yeast extract improves the intensity of the luminescentcolonies on a membrane filter, in comparison to the complete ILM withyeast extract.

I claim:
 1. An enzymatic method for detecting coliform bacteria, in aliquid or liquefied sample comprising the steps of:a) concentrating thebacteria on a membrane filter; b) placing the membrane filter and thebacteria concentrated thereon on a growth medium containing nutrients tosupport propagation of the bacteria and an inducer for inducing a markerenzyme in the course of their growth and metabolism; c) preincubatingthe membrane filter and the bacteria concentrated thereon to formmicrocolonies of these bacteria on the membrane filter and to producesaid marker enzyme; and d) making the microcolonies visible by means ofluminescence, characterized in that said microcolonies are made visibleby an enzyme assay comprising:removing the membrane filter from thegrowth medium after said preincubation step (c); treating themicrocolonies on the membrane filter with a membrane permeabilizer andcontacting them with a chemiluminogenic or fluorogenic substrate forsaid marker enzyme; incubating the microcolonies to allow cleavage ofsaid substrate so as to produce a chemiluminescent or fluorescentproduct; and triggering light emission from the chemiluminescent orfluorescent product produced by the microcolonies.
 2. The methodaccording to claim 1, characterized in that after said preincubationstep (c), the membrane filter is placed on an assay medium containingsaid membrane permeabilizer and said substrate.
 3. The method accordingto claim l, wherein the membrane filter is more hydrophobic thancellulose ester filters.
 4. A method according to claim 1, wherein theinducer is an inducer of β-galactosidase and the fluorogenic substratecomprises a fluorogenic galacto-pyranoside.
 5. A method according toclaim 1, wherein the inducer is an inducer of β-glucuronidase and thefluorogenic substrate comprises a fluorogenic glucuronide.
 6. A methodaccording to claim 1, wherein the inducer is an inducer ofβ-galactosidase and the chemiluminogenic substrate comprises achemiluminogenic galactopyranoside.
 7. A method according to claim 1,wherein the inducer is an inducer of β-glucuronidase and thechemiluminogenic substrate comprises a chemiluminogenic glucuronide. 8.The method according to claim 8, characterized in that thechemiluminescent product produced by cleavage of the chemiluminogenicsubstrate is chemically triggered by an accelerator solution containinga dilute solution of an organic base.
 9. The method according to claim10, wherein the membrane filter is more hydrophobic than cellulose esterfilters.
 10. The method according to claim 1, wherein the membraneperineabilizer comprises polymyxin B and/or colistin methanesulfonate.11. The method according to claim 1, wherein said growth mediumcomprises mineral nutrients, a protein hydrolysate, and a sugar or apolyalcohol.
 12. The growth medium for use in a method according toclaim 1, comprising mineral nutrients, a protein hydrolysate, an inducerfor inducing a marker enzyme in the course of growth and metabolism ofcoliform bacteria, and a sugar or a polyalcohol.
 13. The growth mediumaccording to claim 12, characterized in that said mineral nutrientscomprise monoammonium phosphate, dipotassium hydrogen phosphate, sodiumchloride and magnesium sulfate.
 14. The growth medium according to claim12, comprising inhibitors of non-coliform bacteria.
 15. A methodaccording to claim 1, characterized in that said inducer is an inducerof β-galactosidase and detects faecal or total coliform bacteria.
 16. Amethod according to claim 1, characterized in that said inducer is aninducer of β-glucuronidase and detects E. coli.
 17. A method accordingto claim 7, wherein the chemiluminescent product produced by cleavage ofthe chemiluminogenic substrate is chemically triggered by an acceleratorsolution containing a dilute solution of an organic base.
 18. The methodaccording to claim 17, wherein the membrane filter is more hydrophobicthan cellulose ester filters and said accelerator solution consistsmainly of a dilute solution of said organic base only.
 19. A methodaccording to claim 15, wherein the β-galactosidase inducer is a compoundselected from the group consisting ofisopropyl-β-D-thiogalactopyranoside and meliboise.
 20. A methodaccording to claim 16, wherein the β-glucuronidase inducer is a compoundselected from the group consisting of isopropyl-β-D-thio-glucuronide,pnitrophenyl-β-D-glucuronide, p-nitrophenyl-β-D-glucuronide ando-nitro-phenyl-β-D-glucuronide and methyl-β-D-glucuronide.
 21. Themethod according to claim 3, wherein the membrane filter comprisespolyvinylidenedifluoride (PVDF).
 22. The method according claim 4,wherein the fluorogenic galacto-pyranoside is aumbelliferyl-β-D-galactopyranoside.
 23. The method according to claim22, wherein the umbelliferyl-β-D-galactopyranoside is4-trifluoromethyl-umbelliferyl-β-D-galactopyranoside (TFMU-gal).
 24. Themethod according to claim 5, wherein the fluorogenic glucuronide is anumbelliferyl-β-D-glucuronide.
 25. The method according to claim 24,wherein the umbelliferyl-β-D-glucuronide is4ethyl-umbelliferyl-β-D-glucuronide (TFMUG).
 26. The method according toclaim 6, wherein the chemiluminogenic galactopyranoside is3-(4-methoxyspiro(1,2-dioxetane-3,2'-tri-cyclo(3.3.1.1³.7)decan)-4-yl)phenyl-β-D-galactopyranoside (AMPGD) or aderivative thereof.
 27. The method according to claim 26, wherein thederivative of 4-methoxyspiro(1,2-dioxetane-3,2'-tri-cyclo(3.3.1.1³.7)decan)-4-yl)phenyl-β-D-galactopyranoside is a chloroderivative.
 28. The method according to claim 7, wherein thechemiluminogenic glucuronide is(3-(4methoxyspiro-(1,2-dioxetane-3,2'-(5'-chloro)-tricyclo(3.3.1.1³.7)decan)-4-yl)phenyl)-β-D-glucuronideor a derivative thereof.
 29. The method according to claim 8, whereinthe organic base comprises piperidine.
 30. The method according to claim9, wherein the membrane filter comprises polyvinylidene-difluoride(PVDF) and said accelerator solution consists mainly of a dilutesolution of said organic base.
 31. The method according to claim 30,wherein the organic base comprises piperidine.
 32. The method accordingto claim 11, wherein said growth medium comprises mineral nutrients, theprotein hydrolysate tryptone, and either the sugar maltose or thepolyalcohol mannitol.
 33. A growth medium according to claim 12,comprising mineral nutrients, the protein hydrolysate tryptone, eitheran inducer of β-galactosidase or an inducer of β-glucuronidase, andeither the sugar maltose or the polyalcohol mannitol.
 34. A growthmedium according to claim 14, wherein said inhibitors of non-coliformbacteria are selected from the group consisting of sodium dodecylsulfate, bile salts and ccfsulodin.
 35. A method according to claim 17,wherein the organic base comprises piperidine.
 36. A method according toclaim 18, wherein the membrane filter comprises polyvinylidenedifluoride(PVDF) and said organic base comprises piperidine.